While conventional reversed-phase columns (e.g. C18) are most widely used for small molecule separations, some drawbacks impede their use in certain applications, such as peak tailing of basic molecules at pH7, due to the undesired interactions between the protonated basic molecules and negatively charged underivatized surface silanols (Si—OH) groups. Recent advances in silica synthesis and bonding technology provide solutions to minimize base tailing by using high-purity silica, high surface coverage, and exhaustive end-capping. However, the resulting stationary phases are usually incompatible with highly aqueous mobile phases due to “phase collapse” or “de-wetting.”
Polar-embedded reversed-phase materials can improve the peak shape of basic analytes and make resulting columns fully operational in highly aqueous environment. These phases are primarily hydrophobic but have hydrophilic groups incorporated near the silica surface. The commonly used polar groups are amide, sulfonamide, urea, ether and carbamate functionalities. Two approaches have been used to make such materials. The first reported polar-embedded phase was prepared by a two-step surface modification method. In Step One, silica particles were modified with an aminopropyl silane. In Step Two, the surface amino groups were treated with an alkyl acyl chloride to form an amide linkage between the alkyl chain and the silica surface. The main drawback of this approach is that some un-reacted residual amino groups are always present in the final product, resulting in undesirable chromatographic properties for acidic molecules. The second generation of polar-embedded phases was prepared using a one-step surface modification approach: a silane ligand that contained both alkyl chain and embedded polar group was synthesized first before being bonded to silica particles. While this approach yields an “anion-exchange free” surface, the cost for making such special silane ligand is relatively high, and a subsequent end-capping step to minimize the presence of surface silanol groups is often required.
Another approach to obtain “aqueous-compatibility” reversed-phase stationary phases is to end-cap the reversed-phase surface with a hydrophilic end-capping reagent. In this case, two different silane reagents are needed. In addition, the hydrolytic stability of hydrophilic end-capping group is usually inferior to the reversed-phase ligand, resulting in selectivity drift throughout its lifetime.